First, though, a few general remarks on developmental stages. In some ways, these are somewhat arbitrary: development is an ongoing process, a real continuum, and what we’re doing is picking recognizable moments where we think we see real transitions and highlighting those as significant markers. They can be somewhat fuzzy, although in early development in particular, when the organism is simple, we can find discrete events that we can easily label. For an example of a developmental stage with which you’re all familiar and which often isn’t so discrete, try adolescence. It’s definitely a real stage, but you’ll also have a difficult time naming exactly when yours began and ended. What we typically have to do is pick some characteristic event imbedded in a series of changes, and say that is the start — first menses in girls, for instance, or the appearance of pubic hair or other secondary sexual characteristics.
Delimiting embryonic developmental events have some of the same problems. Gastrulation is said to begin when cells start moving into the interior of the embryo, but of course from a molecular perspective, the process begins earlier, when axis specification establishes the site of inward movement, when cells begin to express particular patterns of genes, and so forth. We’re more and more beginning to see developmental stages defined in terms of which genes are active, to complement the traditional staging by what morphological features are present.
One of the more useful tools in an embryologist’s arsenal is a good staging series for the animal being studied, as it becomes a reference work for keeping track of the progress of experimental animals. I’ve described before a staging series for the bat, Carollia perspicillata, which is often one of the first things the biologist needs to develop before proceeding; I much more often use the zebrafish staging series, although at this point I’ve actually got it mostly memorized. The utility of this sort of information is that now, when I get an embryo out of the incubator, I can look at it and accurately judge how old it is, what it has already done, and predict what’s going to happen next. If I see the beginnings of a tailbud, and count that it has about 14 or 15 somites, I know that in about an hour the first motoneurons will begin to send axons out of the spinal cord, and that it will begin twitching. It also means I can do reproducible experiments: if I want to expose embryos to a drug as soon as gastrulation begins, I can collect a set, observe them to get their age, and schedule the treatments fairly precisely.
You may notice that working staging series, like the zebrafish series, are more detailed than most people know. We can often identify an embryo’s age to within a quarter hour
The blastula stage: In humans, this stage runs from right after fertilization to 14 days after fertilization; in zebrafish, the time is measured in hours, between 0 and 5.25 hours. This period is often subdivided. In zebrafish, for instance, the earliest part is called cleavage, and the term blastula proper is reserved for the time when the mass of cells have flattened into a sheet. In humans, the time around 3 days when the dividing cells form a hollow ball called the blastocyst is regarded as a landmark.
The blastula is a period of proliferation, when cells are dividing rapidly to generate a mass of raw material, and when subtle but essential molecular events, like axis formation, are occurring.
The gastrula stage: gastrulation begins when cells begin to migrate into the interior of the animal to generate the three germ layers, endoderm, mesoderm, and ectoderm. This event begins at about 14 days in humans and proceeds through approximately the 21st day. In zebrafish, it occurs between 5.25 and 10.33 hours.
The neurula stage: during neurulation, the nervous system begins to form first as a flattened sheet, and then as a tube as the sheet rolls up. It ends when the tube is completely closed at the anterior and posterior ends. In humans, this occurs between 19 and 27 days; notice that it overlaps with gastrulation. Even before gastrulation is over, the anterior end of the animal can begin forming the neural tube. In zebrafish, the first neurons are born around 9 hours; the beginning and end of neurulation are less obvious than they are in humans, though, because the formation of the tube is much less obvious, with tightly adherent cells condensing into a thickened, solid rod.
The pharyngula stage: the pharyngula is a vertebrate embryo that has assembled the outlines of the body plan. It has the key features of vertebrate morphology — a post-anal tail, a notochord, a dorsal neural tube, a segmented musculature, and an array of branchial arches (“gill” arches). The major organs have begun to form. It is the stage at which the outline or the framework of the whole body is present in soft focus, with a great deal of refinement and growth ahead of it. It’s the stage at which vertebrate embryos most resemble one another, and is also called the phylotypic stage, to reflect the unity of the phylum.
This stage can be seen in humans between 25 and 30 days after fertilization, and in zebrafish at 24-48 hours.
Here are a pair of representative pharyngulas, a bat (left) and a dolphin (right), two animals whose adult lives are about as radically different as you’ll find in the Mammalia. All of the pieces of a vertebrate are there in rudimentary form, organized in roughly the same layout — both have have pharyngeal arches (the strange blobs and bumps at the head end, near the top of the pictures), both have segmented body musculature (not easily seen at this resolution), they have tails, they have notochords inside. There are also differences, but they’re relatively minor — the degree of flexure of the head, the coiling of the tail, and there are probably quantitative differences in things like the number of segments (the colors, though, are entirely a product of differences in the photography).
Michael Richardson has done a wonderful photographic survey of pharyngula-stage embryos. Here are a few of the animals he has sampled, with all of the pharyngulas in the top row, followed below by later stages of the same species, so you can see how they develop over time. Again, you’ll immediately notice differences—the shape of the head, the curvature of the whole embryo, or the presence of bits of the extraembryonic tissues. What jumps out to the eye of the embryologist, though is that at the pharyngula stage the same elements of morphology are present in about the same relationship to one another. There is a unity to organization at this time that shouldn’t be ignored because there are also little differences here and there.
In particular, the similarities at the pharyngula stage loom large in comparison to the major developmental changes that follow.
Not shown in that picture are earlier stages, like the gastrula and neurula, where differences are also greater. Gastrula stage embryos can be shaped like a disc, a cup, or a ball. Cells can move inward at a single lip of tissue or along a furrow; they can move as contiguous sheets or as delaminated, single cells. The gastrulating embryo can be draped across a gigantic yolk cell that dwarfs it, or there may be no yolk at all.
What this means is that vertebrate embryos of different species resemble each other most at the pharyngula stage. Very early embryos may differ greatly, but they all converge on a broadly similar form, the pharyngula, and then diverge into the diversity of adult forms. Widely different eggs construct relatively similar embryos at a transient period called the phylotypic stage, only to have them subsequently differ again. This pattern is often called the developmental hourglass, where the narrow waist of the hourglass represents the pharyngula.
The best explanation for this pattern comes from Rudy Raff, and is diagrammed in the chart above. The way to define a robust animal form is to specify the general layout at an early stage, when the embryo is still small and relatively simple. At this point, global interactions dominate—molecules are working as gradients or chains of reactions across the whole of the embryo to stake out the general body plan. These gene interactions may, for instance, define a particular region as the place to form an ear, or a forelimb, and they have to do this relative to other regions; mixing up where to put an arm and where to put an ear would be a catastrophic error, so these surveying exercises in whole animal topography tend to be well conserved. On the other hand, once the position of the forelimb has been staked out, development can proceed more locally and autonomously; the genetic program to elaborate a forelimb does not need to consult the genetic program to build an ear in order to proceed. This local independence makes it easier for evolution to sculpt later events than events during the pharyngula stage. The forelimb program, for example, can acquire novel refinements that can spell the difference between making a dolphin’s flipper and a bat’s wing without disturbing other important morphogenetic processes in the body plan.
That’s really the answer to understanding what the pharyngula stage is all about. It’s that point in time when the relationships between the major parts of the body are established, which makes it interesting from both a developmental point of view — it’s the starting point for organogenesis — and from an evolutionary point of view — it’s a common core foundation for evolved differences in the adult morphology of species. It’s also cool enough that when I was casting about for a name for a blog that would discuss both development and evolution, it was a natural.